Anja U. Bräuer

An impaired neocortical Ih is associated with enhanced excitability and absence epilepsy.

Neuronal subthreshold excitability and firing behaviour are markedly influenced by the activation and deactivation of the somato‐dendritic hyperpolarization‐activated cation current (Ih). Here, we evaluated possible contributions of Ih to hyperexcitability in an animal model of absence seizures (WAG/Rij rats). We investigated pyramidal neurons of the somatosensory neocortex, the site of generation of spike–wave discharges. Ih‐mediated functions in neurons from WAG/Rij rats, Wistar rats (sharing the same genetic background with WAG/Rij, but less epilepsy‐prone) and ACI rats (an inbred strain, virtually free of seizures) were compared. We complemented whole‐cell recordings from layer 2–3 pyramidal neurons with immunohistochemistry, Western blot and RT‐PCR analysis of the h‐channel subunits HCN1–4. The fast component of Ih activation in WAG/Rij neurons was significantly reduced (50% reduction in the h‐current density) and four times slower than in neurons from nonepileptic Wistar or ACI rats. The results showing decreases in currents corresponded to a 34% reduction in HCN1 protein in the WAG/Rij compared to the Wistar neocortex, but HCN1 mRNA showed stable expression. The other three Ih subunit mRNAs and proteins (HCN2–4) were not affected. The alterations in Ih magnitude and kinetics of gating in WAG/Rij neurons may contribute to augmented excitatory postsynaptic potentials, the increase in their temporal summation and the facilitation of burst firing of these neurons because each of these effects could be mimicked by the selective Ih antagonist ZD 7288. We suggest that the deficit in Ih‐mediated functions may contribute to the development and onset of spontaneously occurring hyperexcitability in a rat model of absence seizures.

Neuronal 3′,3,5-Triiodothyronine (T3) Uptake and Behavioral Phenotype of Mice Deficient in Mct8, the Neuronal T3 Transporter Mutated in Allan–Herndon–Dudley Syndrome

Thyroid hormone transport into cells requires plasma membrane transport proteins. Mutations in one of these, monocarboxylate transporter 8 (MCT8), have been identified as underlying cause for the Allan–Herndon–Dudley syndrome, an X-linked mental retardation in which the patients also present with abnormally high 3′,3,5-triiodothyronine (T3) plasma levels. Mice deficient in Mct8 replicate the thyroid hormone abnormalities observed in the human condition. However, no neurological deficits have been described in mice lacking Mct8. Therefore, we subjected Mct8-deficient mice to a comprehensive immunohistochemical, neurological, and behavioral screen. Several behavioral abnormalities were found in the mutants. Interestingly, some of these behavioral changes are compatible with hypothyroidism, whereas others rather indicate hyperthyroidism. We thus hypothesized that neurons exclusively dependent on Mct8 are in a hypothyroid state, whereas neurons expressing other T3 transporters become hyperthyroid, if they are exposed directly to the high plasma T3. The majority of T3 uptake in primary cortical neurons is mediated by Mct8, but pharmacological inhibition suggested functional expression of additional T3 transporter classes. mRNAs encoding six T3 transporters, including L-type amino acid transporters (LATs), were coexpressed with Mct8 in isolated neurons. We then demonstrated Lat2 expression in cultured neurons and throughout murine brain development. In contrast, LAT2 is expressed in microglia in the developing human brain during gestation, but not in neurons. We suggest that lack of functional complementation by alternative thyroid hormone transporters in developing human neurons precipitates the devastating neurodevelopmental phenotype in MCT8-deficient patients, whereas Mct8-deficient mouse neurons are functionally complemented by other transporters, for possibly Lat2.

Selenium deficiency increases susceptibility to glutamate-induced excitotoxicity.

Excitotoxic brain lesions, such as stroke and epilepsy, lead to increasing destruction of neurons hours after the insult. The deadly cascade of events involves detrimental actions by free radicals and the activation of proapoptotic transcription factors, which finally result in neuronal destruction. Here, we provide direct evidence that the nutritionally essential trace element selenium has a pivotal role in neuronal susceptibility to excitotoxic lesions. First, we observed in neuronal cell cultures that addition of selenium in the form of selenite within the physiological range protects against excitotoxic insults and even attenuates primary damage. The neuroprotective effect of selenium is not directly mediated via antioxidative effects of selenite but requires de novo protein synthesis. Gel shift analysis demonstrates that this effect is connected to the inhibition of glutamate‐induced NF‐κB and AP‐1 activation. Furthermore, we provide evidence that selenium deficiency in vivo results in a massive increase in susceptibility to kainate‐induced seizures and cell loss. These findings indicate the importance of selenium for prevention and therapy of excitotoxic brain damage.

Inherited cortical HCN1 channel loss amplifies dendritic calcium electrogenesis and burst firing in a rat absence epilepsy model.

While idiopathic generalized epilepsies are thought to evolve from temporal highly synchronized oscillations between thalamic and cortical networks, their cellular basis remains poorly understood. Here we show in a genetic rat model of absence epilepsy (WAG/Rij) that a rapid decline in expression of hyperpolarization‐activated cyclic‐nucleotide gated (HCN1) channels (Ih) precedes the onset of seizures, suggesting that the loss of HCN1 channel expression is inherited rather than acquired. Loss of HCN1 occurs primarily in the apical dendrites of layer 5 pyramidal neurons in the cortex, leading to a spatially uniform 2‐fold reduction in dendritic HCN current throughout the entire somato‐dendritic axis. Dual whole‐cell recordings from the soma and apical dendrites demonstrate that loss of HCN1 increases somato‐dendritic coupling and significantly reduces the frequency threshold for generation of dendritic Ca2+ spikes by backpropagating action potentials. As a result of increased dendritic Ca2+ electrogenesis a large population of WAG/Rij layer 5 neurons showed intrinsic high‐frequency burst firing. Using morphologically realistic models of layer 5 pyramidal neurons from control Wistar and WAG/Rij animals we show that the experimentally observed loss of dendritic Ih recruits dendritic Ca2+ channels to amplify action potential‐triggered dendritic Ca2+ spikes and increase burst firing. Thus, loss of function of dendritic HCN1 channels in layer 5 pyramidal neurons provides a somato‐dendritic mechanism for increasing the synchronization of cortical output, and is therefore likely to play an important role in the generation of absence seizures.

A new phospholipid phosphatase, PRG-1, is involved in axon growth and regenerative sprouting

Outgrowth of axons in the central nervous system is governed by specific molecular cues. Molecules detected so far act as ligands that bind to specific receptors. Here, we report a new membrane-associated lipid phosphate phosphatase that we have named plasticity-related gene 1 (PRG-1), which facilitates axonal outgrowth during development and regenerative sprouting. PRG-1 is specifically expressed in neurons and is located in the membranes of outgrowing axons. There, it acts as an ecto-enzyme and attenuates phospholipid-induced axon collapse in neurons and facilitates outgrowth in the hippocampus. Thus, we propose a novel mechanism by which axons are able to control phospholipid-mediated signaling and overcome the growth-inhibiting, phospholipid-rich environment of the extracellular space.

Autotaxin (NPP-2) in the brain: cell type-specific expression and regulation during development and after neurotrauma.

Abstract.Autotaxin is a secreted cell motility-stimulating exo-phosphodiesterase with lysophospholipase D activity that generates bioactive lysophosphatidic acid. Lysophosphatidic acid has been implicated in various neural cell functions such as neurite remodeling, demyelination, survival and inhibition of axon growth. Here, we report on the in vivo expression of autotaxin in the brain during development and following neurotrauma. We found that autotaxin is expressed in the proliferating subventricular and choroid plexus epithelium during embryonic development. After birth, autotaxin is mainly found in white matter areas in the central nervous system. In the adult brain, autotaxin is solely expressed in leptomeningeal cells and oligodendrocyte precursor cells. Following neurotrauma, autotaxin is strongly up-regulated in reactive astrocytes adjacent to the lesion. The present study revealed the cellular distribution of autotaxin in the developing and lesioned brain and implies a function of autotaxin in oligodendrocyte precursor cells and brain injuries.

Synaptic PRG-1 Modulates Excitatory Transmission via Lipid Phosphate-Mediated Signaling

Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.Plasticity related gene-1 (PRG-1) is a brain-specific membrane protein related to lipid phosphate phosphatases, which acts in the hippocampus specifically at the excitatory synapse terminating on glutamatergic neurons. Deletion of prg-1 in mice leads to epileptic seizures and augmentation of EPSCs, but not IPSCs. In utero electroporation of PRG-1 into deficient animals revealed that PRG-1 modulates excitation at the synaptic junction. Mutation of the extracellular domain of PRG-1 crucial for its interaction with lysophosphatidic acid (LPA) abolished the ability to prevent hyperexcitability. As LPA application in vitro induced hyperexcitability in wild-type but not in LPA(2) receptor-deficient animals, and uptake of phospholipids is reduced in PRG-1-deficient neurons, we assessed PRG-1/LPA(2) receptor-deficient animals, and found that the pathophysiology observed in the PRG-1-deficient mice was fully reverted. Thus, we propose PRG-1 as an important player in the modulatory control of hippocampal excitability dependent on presynaptic LPA(2) receptor signaling.

Developmental and cell type-specific expression of thyroid hormone transporters in the mouse brain and in primary brain cells.

Cellular thyroid hormone uptake and efflux are mediated by transmembrane transport proteins. One of these, monocarboxylate transporter 8 (MCT8) is mutated in Allan‐Herndon‐Dudley syndrome, a severe mental retardation associated with abnormal thyroid hormone constellations. Since mice deficient in Mct8 exhibit a milder neurological phenotype than patients, we hypothesized that alternative thyroid hormone transporters may compensate in murine brain cells for the lack of Mct8. Using qPCR, Western Blot, and immunocytochemistry, we investigated the expression of three different thyroid hormone transporters, i.e., Mct8 and L‐type amino acid transporters Lat1 and Lat2, in mouse brain. All three thyroid hormone transporters are expressed from corticogenesis and peak around birth. Primary cultures of neurons and astrocytes express Mct8, Lat1, and Lat2. Microglia specifically expresses Mct10 and Slco4a1 in addition to high levels of Lat2 mRNA and protein. As in vivo, a brain microvascular endothelial cell line expressed Mct8 and Lat1. 158N, an oligodendroglial cell line expressed Mct8 protein, consistent with delayed myelination in MCT8‐deficient patients. Functional T3‐ and T4‐transport assays into primary astrocytes showed KM values of 4.2 and 3.7 μM for T3 and T4. Pharmacological inhibition of L‐type amino acid transporters by BCH and genetic inactivation of Lat2 reduced astrocytic T3 uptake to the same extent. BSP, a broad spectrum inhibitor, including Mct8, reduced T3 uptake further suggesting the cooperative activity of several T3 transporters in astrocytes.

Molecular analysis of Nogo expression in the hippocampus during development and following lesion and seizure

The Nogo gene encodes an integral membrane protein mainly responsible for the neurite inhibition properties of myelin. Here, we analyzed the expression pattern of Nogo-A, Nogo-B, and Nogo-C and Nogo-66 receptor (Ng66R) mRNA during hippocampal development and lesion-induced axonal sprouting. Nogo-A and Nogo-B and Ng66R transcripts preceded the progress of myelination and were highly expressed at postnatal day zero (P0) in all principal hippocampal cell layers, with the exception of dentate granule cells. Only a slight Nogo-C expression was found at P0 in the principal cell layers of the hippocampus. During adulthood, all Nogo splice variants and their receptor were expressed in the neuronal cell layers of the hippocampus, in contrast to the myelin basic protein mRNA expression pattern, which revealed a neuronal source of Nogo gene expression in addition to oligodendrocytes. After hippocampal denervation, the Nogo genes showed an isoform-specific temporal regulation. All Nogo genes were strongly regulated in the hippocampal cell layers, whereas the Ng66R transcripts showed a significant increase in the contralateral cortex. These data could be confirmed on protein levels. Furthermore, Nogo-A expression was up-regulated after kainate-induced seizures. Our data show that neurons express Nogo genes with a clearly distinguishable pattern during development. This expression is further dynamically and isoform-specifically altered after lesioning during the early phase of structural rearrengements. Thus, our results indicate a role for Nogo-A, -B, and -C during development and during the stabilization phase of hippocampal reorganization. Taken together with these data, the finding that neurons in a highly plastic brain region express Nogo genes supports the hypothesis that Nogo may function beyond its known neuronal growth inhibition activity in shaping neuronal circuits.

Neurotractin/kilon promotes neurite outgrowth and is expressed on reactive astrocytes after entorhinal cortex lesion

The IgLON subgroup of the immunoglobulin superfamily consists of four members that are thought to be important in neural cell-cell recognition. Here, we cloned and characterized the murine IgLON subgroup member neurotractin/kilon, in the context of brain development and axonal regeneration. Neurotractin/kilon was found to be upregulated during brain development and is expressed on neurites of primary hippocampal neurons. To elucidate a potential role for neurotractin/kilon during regeneration in the CNS, we performed lesions in the entorhinal cortex, and showed that the expression of neurotractin/kilon is induced on reactive astrocytes. Notably, the expression on reactive astrocytes appears specifically in the denervated outer molecular layer of the dentate gyrus, where regenerative axon sprouting occurs. In vitro assays demonstrated that neurotractin/kilon attracts hippocampal axons in the stripe assay and that astroglial neurotractin/kilon promotes neurite outgrowth. These results suggest a function for neurotractin/kilon as a trans-neural growth-promoting factor for outgrowing axons following hippocampal denervation.